Recombinant proteins as therapeutic modalities have found increasing use in recent years. Numerous recombinant protein-based therapies are in various stages of clinical development. One limitation to widespread clinical application of recombinant protein technology is the difficulty in production of proteins that include two or more coding sequences or domains such that the domains are expressed in the proper ratio with appropriate post-translational processing resulting in production of a functional heterodimeric molecule. A further limitation is the high cost associated with adequate levels of expression for clinical applications.
Chinese Hamster Ovarian (CHO) cells are the most commonly used mammalian cell lines for commercial scale production of glycosylated human proteins. Previous attempts to express a full length recombinant protein with two or more domains or chains (and thus two or more coding sequences or open reading frames (ORFs)) via recombinant DNA technology using a single vector have met with limited success, typically resulting in unequal levels of expression of the two or more domains or chains of the protein or polypeptide and more particularly, a lower level of expression for the second coding sequence. In order to express a fully biological functional protein or polypeptide which has two or more domains or chains from a single vector, equimolar expression of the two or more domains or chains is typically required. Additionally, conventional vectors relying on dual promoter regulation of gene expression are invariably affected by promoter interaction (i.e., promoter interference) which may compromise equimolar or substantially equimolar expression of the genes. Other factors that limit the ability to express two or more coding sequences from a single vector include the packaging limitation of the vector itself. For example, in considering the appropriate vector/coding sequence combination, factors to be considered include: the capacity of the vector (e.g., approx. 4,500 bp for AAV); the duration of expression of the recombinant molecule by vector-transduced cells (e.g., short term expression for adenoviral vectors); the cell types infected by the vector if a viral vector is used; and the desired expression level of the target gene product(s). The requirement for controlled expression of two or more gene products together with the packaging limitations of viral vectors such as adenovirus and AAV restrict the choices with respect to vector construction and systems for expression of a protein or polypeptide which has two or more domains or chains.
The linking of proteins in the form of polyproteins in a single open reading frame is a strategy adopted in the replication of many viruses including picornaviridae. Upon translation, virus-encoded proteinases mediate rapid intramolecular (cis) cleavage of a polyprotein to yield discrete mature protein products. Foot and Mouth Disease viruses (FMDV) are a group within the picornaviridae which express a single, long open reading frame encoding a polyprotein of approximately 225 kD. The full length translation product undergoes rapid intramolecular (cis) cleavage at the C-terminus of a self-processing cleavage site, for example, a 2A site or region, located between the capsid protein precursor (P1-2A) and replicative domains of the polyprotein 2BC and P3, with the cleavage mediated by proteinase-like activity of the 2A region itself (Ryan et al., J. Gen. Virol. 72:2727-2732, 1991); Vakharia et al., J. Virol. 61:3199-3207, 1987). Similar domains have also been characterized from aphthoviridea and cardioviridae of the picornavirus family (Donnelly et al., J. Gen. Virol. 78:13-21, 1997).
In order to express proteins or polypeptides which have two or more domains or chains from a single vector, two or more promoters or an internal ribosome entry site (IRES) sequence are generally used to drive expression of individual genes. The use of two promoters within a single vector can result in low protein expression, e.g., due to promoter interference. When two genes are linked with an IRES sequence, the expression level of the second gene is often significantly lower than the first gene (Furler et al., Gene Therapy 8:864-873, 2001).
There remains a need for improved gene expression systems for production of recombinant proteins and polypeptides, in particular proteins and polypeptides that have two or more domains or chains, such that sufficient expression of a biologically active recombinant protein or polypeptide is achieved at commercially reasonable cost.
The present invention addresses this need by demonstrating the feasibility and use of a single vector or plasmid construct comprising a sequence that encodes a self-processing peptide which results in the expression of functional recombinant proteins and polypeptides.